Background: The TMEM16A chloride channel serves as depolarising mechanism in arterial smooth muscle cells (SMCs). The channel is a suggested target for diseases of impaired vascular tone including stroke, vascular dementia, and (systemic, pulmonary) hypertension [1, 2]; however, the selectivity and mode of action of available TMEM16A modulators are incompletely defined. We define the mode of action of a recently disclosed positive allosteric modulator (Patent WO2021/014168, compound 1) [3] of the TMEM16A channel (PAM_16A) and examine its selectivity alongside that of a series of established TMEM16A inhibitors (Ani9, MONNA and CaCCinhA01).
Methods: Patch-clamp electrophysiology, isometric tension recordings of isolated rat arteries were employed to study the effects of selective pharmacological control of TMEM16A on vascular function. Data are given as mean±SEM alongside the number of independent experiments (n) and number of animals (N).
Results: In the presence of sub-maximal (300 nM) intracellular free Ca2+ concentration [Ca2+]i, PAM_16A activated the heterologously expressed TMEM16A channels at positive and negative potentials (EC50≈3.6±0.5 nM), while producing a minimal effect on the highly homologous TMEM16B channel. PAM_16A did not activate the TMEM16A currents in the absence of intracellular Ca2+ or in the presence of saturating [Ca2+]i (12 µM). Non-stationary noise analysis revealed that PAM_16A caused an increase in channel open probability. Consistently, mutant TMEM16A channels with the intracellular gate constitutively open were much less sensitive to PAM, suggesting that PAM may act as a modifier of TMEM16A channel gating. PAM_16A selectively activated TMEM16A currents in isolated SMCs by 1.3±0.1 fold at +100 mV and 4.4±0.6 fold at -100 mV (N=5, n=17) (p<0.05) and promoted aortic smooth muscle contraction by 1.6±0.8 fold (N=7, n=9) (p<0.05). Unlike PAM_16A and Ani9, a range of other available modulators were found to interfere with endogenous cationic currents in SMCs.
Conclusion: Arterial tone can be finely controlled with TMEM16A modulators higlighting the channel as a posisble novel therapeutic target in a range of disorders involving impaired vascular tone, including stroke, and hypertension.